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International Immunology, Vol. 13, No. 2, 167-179, February 2001
© 2001 Japanese Society for Immunology

Signaling events following chemokine receptor ligation in human dendritic cells at different developmental stages

Katsuaki Sato, Hiroshi Kawasaki1, Hitomi Nagayama, Makoto Enomoto, Chikao Morimoto1, Kenji Tadokoro2, Takeo Juji2 and Tsuneo A. Takahashi

Department of Cell Processing, and
1 Department of Clinical Immunology and AIDS Research Center, Institute of Medical Science, University of Tokyo, 4-6-1 Shirokanedai, Minato-ku, Tokyo 108-8639, Japan
2 Japanese Red Cross Central Blood Center, 4-1-31 Hiroo, Shibuya-ku, Tokyo 150-1211, Japan

Correspondence to: Correspondence to: T. A. Takahashi


   Abstract
Top
 Abstract
Introduction
 Methods
 Results
 Discussion
 References
 
Responsiveness of dendritic cells (DC) to inflammatory CC chemokines is down-regulated during their maturation. We analyzed the mechanism underlying these events. Cell-surface expression of CC chemokine receptor (CCR)-1, -3 and -5 was increased during differentiation of immature DC (iDC) from monocytes. In contrast, these expressions were decreased during development of iDC into mature DC (mDC) to levels similar to those of monocytes. Transcriptional expression of CCR-1, -3 and -5 was increased during differentiation of iDC from monocytes, while the expression was decreased during development of iDC into mDC. Expression of CCR-7 transcript was detected in mDC, but not in monocytes or iDC. Both monocytes and iDC, but not mDC, migrated in response to inflammatory CC chemokines such as regulated on activation normal T cell expressed and secreted (RANTES)/CCL5, whereas mDC, but not monocytes or iDC, migrated to macrophage inflammatory protein (MIP)-3ß/CCL19. Receptor engagement of monocytes or iDC by RANTES (for CCR-1, -3 and -5) resulted in protein tyrosine phosphorylation events including activation of focal adhesion kinase as well as mitogen-activated protein kinase, whereas this stimulation induced little activation of these molecular events in mDC when compared with monocytes or iDC. On the other hand, stimulation with MIP-3ß (for CCR-7) induced tyrosine phosphorylation events in mDC, but not in monocytes or iDC. These results suggest that the down-regulation of cell-surface expression of CCR and of their downstream signaling events may be involved in the reduced chemotaxis of DC to inflammatory CC chemokines during their maturation.

Keywords: chemotaxis, protein tyrosine kinase, signal transduction


   Introduction
Top
 Abstract
 Introduction
Methods
 Results
 Discussion
 References
 
Dendritic cells (DC) are unique professional major antigen-presenting cells (APC) capable of stimulating resting T cells in the primary immune response and are more potent APC than peripheral blood monocytes/macrophages or B cells (1). DC also play major roles in autoimmune diseases, graft rejection, human immunodeficiency virus infection and the generation of T cell-dependent antibodies (1–4). DC capture and process antigen in non-lymphoid tissues, and then migrate to T cell-dependent areas of secondary lymphoid organs through afferent lymph or the blood stream to prime native T cells and initiate immune responses (5,6).

Characterization of DC is difficult because they represent only a small subpopulation that includes interdigitating reticulum cells in lymphoid organs, blood DC, Langerhans cells in the epidermis of the skin and dermal DC (1). Previously, an in vitro culture system revealed that DC originate from CD34+ pluripotent hematopoietic progenitor cells in the bone marrow and cord blood, and from peripheral blood monocytes via myeloid lineage cells in human and murine models (7–16), and that some DC develop from thymic precursors via lymphoid lineage cells in the murine system (17).

Chemokines are crucially involved in inflammatory/ immunological responses via their capacity to recruit selective leukocyte subsets (18). Chemokines have been implicated in the regulation of normal leukocyte recirculation and homing, and also in certain physiological and pathogenic processes, including hematopoiesis, angiogenesis, allergy, autoimmune diseases and viral infectious diseases (18). Chemokines are a group of ~70–90 amino acid structurally related polypeptides, most of which contain four conserved cysteine residues in their primary amino acid sequence (18). There are two major groups: the CC chemokines, in which the two N-terminal cysteines are adjacent, and the CXC chemokines in which the two N-terminal cysteines are separated by a single amino acid.

The specific effects of chemokines on the target cell types are mediated by a family of single-chain, seven-helix membrane-spanning receptors coupled to heterotrimeric guanine nucleotide-binding protein (G protein) (GPCR) (18). Ligand specificities of 15 CC and CXC chemokine receptors (CCR and CXCR respectively) have been identified; 10 of them are specific for CC chemokines (CCR1–10) (18–20) and five of them are specific for CXC chemokines (CXCR1–5) (18–20). In addition, distinct chemokines appear to act on more than one receptor type (18).

Engagement of chemokine receptors by their appropriate ligands (chemokines) induces an elaborate biochemical program that ultimately results in the induction of a variety of functions including cell migration and proliferation (21,22). The biochemical events include GPCR-dependent calcium influx as well as changes in intracellular cAMP levels and phosphoinositol lipid metabolism (21). Activation signals following stimulation by chemokines also involve the initiation of a protein tyrosine kinase (PTK)-dependent pathway including activation of a family of focal adhesion kinase (FAK) proteins, p125FAK and Pyk2, which leads to reorganization of various cytoskeletal proteins including paxillin as well as activation of phosphatidylinositol 3-kinase and Janus kinase (Jak)/signal transducer and activator of transcription (Stat) cascades (21–27). Furthermore, stimulation with chemokines via their respective GPCR leads to the downstream modulation of a family of small G protein-activated cascades, resulting in the activation of a group of mitogen-activated protein kinases (MAPK) (21,22,25,27–32).

MAPK are activated following engagement of a variety of cell-surface receptors via dual tyrosine and threonine phosphorylation (33–36). The various members of the MAPK families differ in their substrate specificity, and are activated by distinct upstream regulators and extracellular stimuli (33–36). Currently, the MAPK family is comprised of three subfamilies, i.e. (i) the extracellular signal-regulated kinase (ERK) subfamily including p42mapk/erk2 and p44mapk/erk1, (ii) the stress-activated protein kinase/c-Jun N-terminal kinase (JNK/SAPK) subfamily including the p46 JNK/SAPK and p54 JNK/SAPK isoforms and their variants, and (iii) the p38mapk subfamily (33–36). Activation of ERK2, JNK/SAPK and p38mapk, whose activities are regulated through their respective upstream regulators including small G proteins (Ras/Rac/Rho), phosphorylate their transcription factor substrates including ElK-1, c-Jun and activating transcription factor (ATF)-2 in response to a variety of extracellular stimuli, and these events regulate cell morphology, migration and proliferation (21,22,27–36). Recently, Pyk2 has been shown to act as a mediator of regulation of MAPK activity in response to various stimuli in several cell types (25,30).

There is increasing interest in the potential role of chemokines and their respective receptors in the biological properties of DC to clarify the mechanism underlying DC-mediated regulation of immune/inflammatory responses. Previous studies have shown that several chemokine receptors are expressed on some DC and their progenitor cells (19,37–47). Recent studies have shown that the chemotactic migratory properties in response to certain chemokines are strictly regulated in the development of DC from their progenitor cells, and these regulatory mechanism have been potentially implicated in mediating the trafficking of DC and their progenitor cells from blood to tissues and then to lymph nodes where they form a close association with T cells in the process of antigen presentation (19,40,41,46,47). However, the mechanism underlying the changes of chemotactic ability in the development of DC from their progenitor cells remains unclear.

In this report, we examined the potential roles of CCR and their downstream signaling cascades in the regulation of chemotactic migratory properties of monocyte-derived DC.


   Methods
Top
 Abstract
 Introduction
 Methods
Results
 Discussion
 References
 
Media and reagents
The medium used throughout was RPMI 1640 supplemented with 2 mM L-glutamine, 50 µg/ml streptomycin, 50 U/ml penicillin and 10% heat-inactivated FCS. Granulocyte macrophage colony stimulating factor (GM-CSF) was kindly provided by Kirin Brewery (Tokyo, Japan). IL-4, tumor necrosis factor (TNF)-{alpha}, regulated on activation normal T cell expressed and secreted (RANTES), macrophage inflammatory protein (MIP)-1{alpha}, eotaxin and MIP-3ß were purchased from PeproTech (London, UK). Horseradish peroxidase (HRP)-conjugated anti-phosphotyrosine (pTyr) mAb (clone RC20) was purchased from Transduction Laboratories (Lexington, KY). Antibodies to CCR-1, CCR-3, CCR-5, Src, p125FAK, Pyk2 and paxillin, which were used for Western blotting and immune complex kinase assay, were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). ERK2, SAPK/JNK and p38mapk immunoblotting kits and their kinase assay kits were purchased from New England Biolabs (Beverly, MA). Genistein and pertussis toxin were purchased from Sigma (St Louis, MO). The preparations of mAb to CCR-1 (IgG1{kappa}, 141-2) and CCR-3 (IgG1{kappa}, 444-11) were described previously (46).

In vitro generation and culture of human DC
DC were generated from peripheral blood mononuclear cells (PBMC) as described previously (8–10) with some modifications (46–49). Briefly, PBMC were obtained from 30 ml of leukocyte-enriched buffy coat from healthy donors by centrifugation with Ficoll-Hypaque (Pharmacia Biotech, Uppsala, Sweden) and the light density fraction from the 42.5–50% interface was recovered. The cells were resuspended in culture medium and allowed to adhere to six-well plates (Costar, Cambridge, MA). After 2 h at 37°C, non-adherent cells were removed and adherent cells were collected, and these cells were subsequently negative selected with anti-CD2 mAb-conjugated immunomagnetic beads (Dynal, Oslo, Norway) and anti-CD19 mAb-conjugated immunomagnetic beads (Dynal) to deplete CD2+ cells (T cells and NK cells) and CD19+ cells (B cells) according to the manufacturer's instructions. The resulting cells (>95% CD14+ cells) were used as monocytes and cultured in 3 ml of medium supplemented with GM-CSF (50 ng/ml) and IL-4 (50 ng/ml). After 7 days of culture, immature DC (iDC) were harvested. For preparation of mature DC (mDC) from iDC, the cells were subsequently cultured with TNF-{alpha} (50 ng/ml) for another 4 days. These cell populations exhibited a typical mature phenotype of DC as described previously (46–49). The cell differentiation was monitored by light microscopy and the resulting cells were used for subsequent experiments. The viabilities of monocytes, iDC and mDC were >95% as assessed by propidium iodide (PI; Sigma) staining (Becton Dickinson).

Flow cytometry
For surface marker analysis, monocytes, iDC or mDC were cultured with the following mAb conjugated to FITC or PE for direct fluorescence: CD1a (T6-RD1) and CD83 (IM2218) (all from Coulter Immunology, Hialeah, FL), CD14 (Leu-M3) (Becton Dickinson), and CCR-5 (2D7) (PharMingen, San Diego, CA). The cells were also stained with the corresponding FITCor PE-conjugated isotype-matched control mAb (all from Becton Dickinson). In indirect staining, the cells were incubated with biotin-conjugated anti-CCR-1 mAb (141-2) or biotin-conjugated anti-CCR-3 mAb (444-11) for 30 min at 4°C, washed twice with cold PBS and subsequently stained with FITC-conjugated avidin (Becton Dickinson) for 30 min at 4°C. Thereafter, the cells were washed twice and suspended in PBS containing 0.2 µg/ml PI to exclude dead cells. Analysis of fluorescence staining was performed with a FACSCalibur flow cytometer (Becton Dickinson) and CellQuest Software. The expression levels of CCR-1 and -3 were also confirmed by flow cytometry with anti-CCR-1 mAb (R & D Systems, Minneapolis, MN) and anti-CCR-3 mAb (R & D Systems).

Semiquantitative RT-PCR
RNA from each sample (5x106) was isolated using Trizol LS reagent (Gibco/BRL, Gaithersburg, MD). The first-strand cDNA kit (SuperScript preamplication system; Gibco/BRL) was used to make cDNA (20 µl) from 5 µg of each RNA. Amplification of each cDNA (1 µl) was performed with a SuperTaq Premix kit (Sawady, Tokyo, Japan) using specific primers as follows: CCR-1 (47), 5'-TCC TCA CGA AAG CCT ACG AGA GTG GAA GC-3' and 5'-CCA CGG AGA GGA AGG GGA GCC ATT TAA C-3'; CCR-3 (47), 5'-TTT GGT GTC ATC ACC AGC AT-3' and 5'-TCA TGC AGC AGT GGG AGT AGG-3'; CCR-5 (47), 5'-GGT GGA ACA AGA TGG ATT AT-3' and 5'-CAT GTG CAC AAC TCT GAC TG-3'; CCR-7 (47), 5'-GAT TAC ATC GGA GAC AAC ACC-3' and 5'-TAG TCC AGG CAG AAG AGT CG-3'. Specific primers for ß-actin (Toyobo, Osaka, Japan) were also used for amplification. The reaction mixture was subjected to 35 cycles of PCR with the following conditions: CCR-1; 95°C for 30 s, 55°C for 30 s and 72°C for 1 min. CCR-3 and -5; 94°C for 1 min, 55°C for 1 min and 72°C for 1 min. CCR-7; 94°C for 1 min, 61.5°C for 2 min and 72°C for 3 min. PCR products were analyzed by electrophoresis through 2% agarose gels and visualized under UV light after ethidium bromide staining.

Assay for chemotaxis
The in vitro migration of monocytes, iDC or mDC in response to CC chemokines was assessed in a Transwell cell culture chamber (Costar) as described previously (46,47,49). In brief, 8.0 µm pore size polycarbonate filters were precoated with 5 µg of gelatin (Wako, Osaka, Japan) in a volume of 50 µl on the lower surface and dried overnight at room temperature. The coated filters were washed in PBS and then dried immediately before use. The cells were pretreated with or without genistein (10 µM) or pertussis toxin (100 ng/ml) for 30 min at 37°C and 100 µl of the cell suspension (106) was added to the upper compartment of the chamber. RANTES, MIP-1{alpha}, eotaxin or MIP-3ß (1–100 ng/ml) diluted in serum-free culture medium (600 µl) was loaded in the lower compartment. After a 2 h incubation, the filters were fixed with methanol and stained with hematoxylin & eosin (all from Wako). The cells on the upper surface of the filters were removed by wiping with cotton swabs. The cells that had migrated to various areas of the lower surface were manually counted under a microscope at a magnification of x200 and each assay was performed in triplicate. The data is expressed as no. of migrated cells/field.

Binding assay
Binding of 125I-labeled chemokine to targeted cells was assayed according to previous reports (50,51) with some modifications. In brief, monocytes, iDC or mDC (2x106/sample) were resuspended in 200 µl of binding medium (RPMI 1640/1% BSA) and incubated for 1 h at room temperature with 125I-labeled RANTES (~0.1 nM) (sp. act. 2200 Ci/mmol; NEN Life Science Products, Boston, MA) in the presence or absence of increasing concentrations (0.001–1000 nM) of competitive unlabeled (cold) RANTES. The cells were then spun (12,000 r.p.m., 1 min) through an 800 µl cushion of 10% (w/v) sucrose in PBS. The pellet was dried and then counted using a Wizard 1470 automatic {gamma}-counter (Pharmacia Biotech). Non-specific binding was determined in the presence of 1 µM unlabeled ligand. The binding data was analyzed using Ligand to determine the half-maximal inhibitory concentrations (IC50) values and the specific binding sites (50,51).

Western blotting and immune complex kinase assay
Monocytes, iDC or mDC were starved in serum-free medium for 16 h at 37°C and subsequently kept for 4 h on ice to reduce the basal levels of tyrosine phosphorylation of intracellular proteins (49,52). The cells (4x106) were untreated or stimulated with RANTES or MIP-3ß (10 ng/ml) for 5 min at 37°C, and washed twice in cold PBS, resuspended in 100 µl of lysis buffer (1% NP-40, 20 mM Tris–HCl, pH 8.0, 137 mM NaCl, 10% glycerol, 2 mM EDTA, 10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM PMSF and 1 mM sodium orthovanadate) and total cell lysates were obtained. To prepare immunoprecipitated samples, total cell lysates (107 cells) were immunoprecipitated with an antibody to CCR-1, CCR-3, CCR-5, Src, p125FAK, Pyk2 or paxillin, immunocomplex were collected by Protein G–Sepharose 4 fast flow (Pharmacia Biotech) and washed 3 times with lysis buffer. Total cell lysates or the immunoprecipitates sample were suspended in 2xSDS sample buffer (313 mM Tris–HCl, pH 6.8, 10% SDS, 2% 2-mercaptoethanol, 50% glycerol and 0.01% bromophenol blue) and heated for 3 min at 95°C. The samples were fractionated by 10 or 12% SDS–PAGE, transferred onto PVDF membranes (Millipore, Bedford, MA) and probed with HRP-conjugated anti-pTyr mAb. Blots were visualized by enhanced chemiluminescence (ECL) (New England Biolabs). To ensure similar amounts of respective proteins in each sample, the same membrane was stripped off, reprobed with the stated antibodies and developed with HRP-conjugated secondary antibodies (Santa Cruz Biotechnology) by ECL. For immune complex kinase assay, the immunoprecipitates with antibodies to p125FAK or Pyk2 from total cell lysates (107 cells) was washed 3 times with lysis buffer and twice with kinase buffer before resuspending in 20 µl of kinase buffer containing 10 µM ATP (New England Biolabs), 5 µCi [{gamma}-32P]ATP (NEN Life Science Products) and 5 µg enolase (Sigma). An autophosphorylation assay was also performed by incubating the immune complex in a kinase buffer containing 10 µM ATP (New England Biolabs) and 5 µCi [{gamma}-32P]ATP. The mixtures were incubated at 30°C for 15 min, and the reactions were terminated by adding 2xSDS sample buffer followed by heated for 3 min at 95°C, separated by 10% SDS–PAGE, transferred onto PVDF membranes and subjected to autoradiography. Immunoblotting and in vitro kinase assay of ERK2, SAPK/JNK or p38mapk were performed with their respective kits according to the manufacturer's instructions manual (49,52).


   Results
Top
 Abstract
 Introduction
 Methods
 Results
Discussion
 References
 
Expression levels of CCR in monocytes, iDC and mDC
We (46,47) have previously reported that human monocyte-derived DC expressed CCR-1, -3 and -5 on their cell surface, and exhibited chemotaxis in response to their respective ligands. To examine the relationship between the cell-surface expression levels of CCR-1, -3 and -5 and the developmental stage of human peripheral blood monocyte-derived DC, flow cytometric analysis of monocytes, iDC and mDC was performed using mAb to respective CCR. We established iDC (<1% CD14+ cells, >95% CD1a+ cells, <1% CD83+ cells) from monocytes (>95% CD14+ cells, <1% CD1a+ cells, <1% CD83+ cells) using GM-CSF (50 ng/ml) and IL-4 (50 ng/ml) for 7 days and mDC (<1% CD14+ cells, >95% CD1a+ cells, >95% CD83+ cells) were obtained by culturing iDC with TNF-{alpha} (50 ng/ml) for another 4 days (Fig. 1AGo), and these results were consistent with previous reports (9,47,49). We also observed that the development of iDC into mDC was accompanied with up-regulation of CD11c, CD40, CD80, CD86 and HLA-DR (48–50). Cell-surface expression levels of CCR-1, -3 and -5 were elevated in the differentiation of monocytes into iDC (Fig. 1BGo). On the other hand, these levels were decreased in the development of iDC into mDC and the levels of these CCR in mDC were similar to those of monocytes (Fig. 1BGo). We also observed that transcriptional expression of CCR-1, -3 and -5 was increased in the differentiation of monocytes into iDC, whereas the expression was significantly decreased during the development of iDC into mDC (Fig. 1CGo).




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  		Fig. 1. Expression of CCRs in monocytes, iDC and mDC. (A) Development of monocyte-derived DC. Monocytes, iDC and mDC were stained with indicated mAb or isotype-matched controls. Cell-surface expression was analyzed by FACS. Data are represented by a dot-plot. (B) Cell-surface expression levels of CCR in monocytes, iDC and mDC. Monocytes, iDC and mDC were stained with indicated mAb or isotype-matched controls. Cell-surface expression was analyzed by FACS. Data are represented by histograms: mAb (thick lines) and isotype-matched controls (thin lines). The values shown in the flow cytometry profiles are the mean fluorescence intensity indexes and the values of the background FITC staining were <8. (C) Transcriptional expressions of CCR in monocytes, iDC and mDC. RNA was extracted from monocytes (1), iDC (2) and mDC (3), and the expression of CCR-1, -3, -5 and -7 mRNA were determined by semiquantitative RT-PCR. PCR products for CCR-1 (440 bp), CCR-3 (444 bp), CCR-5 (1117 bp), CCR-7 (1067 bp) and ß-actin (645 bp) are shown. The results of RT-PCR for ß-actin demonstrate the loading of equal amounts of DNA on the gel. The results are representative of 10 experiments performed with similar results.

 
Previous studies have shown that the CCR-7 transcript is exclusively expressed in mDC and these cells migrate to MIP-3ß/CCL19 (20) via CCR-7 (19,40,41,47). As shown in Fig. 1Go(C), although both monocytes and iDC did not express CCR-7 transcript, mDC exhibited its transcriptional expression. We also observed similar expression levels of CCR-1, -3, -5 and -7 in lipopolysaccharide (LPS)-, anti-CD40 mAb- and monocyte-conditioned medium-induced mDC to TNF-{alpha}-induced mDC (data not shown).

The chemotactic migratory capacities of monocytes, iDC and mDC in response to CC chemokines
To elucidate the relationship between the chemotactic migratory capacity via CCR and developmental stage of monocyte-derived DC, we examined the abilities of monocytes, iDC and mDC to migrate in response to RANTES/CCL5 (for CCR-1, -3 and -5), MIP-1{alpha}/CCL3 (for CCR-1 and CCR-5), eotaxin/CCL11 (for CCR-3) or MIP-3ß/CCL19 (for CCR-7) (20). As shown in Fig. 2Go(A–C), the chemotactic migratory responses to inflammatory chemokines such as RANTES, MIP-1{alpha} and eotaxin were increased during development of iDC from monocytes, whereas these properties were significantly decreased in the differentiation of iDC into mDC, and these results are consistent with previous reports (47). On the other hand, both monocytes and iDC failed to migrate in response to MIP-3ß, while mDC exhibited this migration (Fig. 2DGo).



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  		Fig. 2. Migration of monocytes, iDC and in mDC in response to CC chemokines. The cells (106) were seeded in upper chambers with filters which were precoated on the lower surface with 5 µg of gelatin. RANTES (A), MIP-1{alpha} (B), eotaxin (C) and MIP-3ß (D) were added to lower chambers. After a 2 h incubation, the cells that migrated to the lower surface were microscopically counted. The results are representative of 10 experiments with similar results.

 
Effect of genistein and pertusis toxin on chemotaxis of monocytes, iDC and mDC in response to CC chemokines
To examine the molecular mechanism underlying the chemotaxis of monocytes, iDC and mDC to CC chemokines, we examined the effect of genistein and pertusis toxin on the chemotaxis of these cells to RANTES and MIP-3ß. As shown in Fig. 3Go(A–C), the chemotactic migratory responses of these cells to RANTES and MIP-3ß were significantly suppressed by genistein and pertusis toxin. These results indicate that the chemotaxis of monocytes, iDC and mDC to these CC chemokines is mediated by PTK and Gi protein-coupled receptor.



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  		Fig. 3. Effect of genistein and pertussis toxin on migration of monocytes, iDC and mDC in response to CC chemokines. Monocytes (A), iDC (B) and mDC (C) were pretreated with or without genistein (10 µM) or pertussis toxin (100 ng/ml) for 30 min at 37°C, and the cells (106) were seeded in upper chambers with filters which were precoated on the lower surface with 5 µg of gelatin. RANTES or MIP-3ß (100 ng/ml) used as chemoattractant was added to lower chambers. After a 2 h incubation, the cells that migrated to the lower surface were microscopically counted. The results are representative of 10 experiments with similar results.

 
Binding of [125I]RANTES to monocytes, iDC and mDC
To examine the change of the binding activities of monocyte-derived DC to RANTES, monocytes, iDC and mDC were incubated with 125I-labeled RANTES in the presence or absence of increasing concentrations (000.1–1000 nM) of unlabeled RANTES, and their specific binding counted. Figure 4Go shows that unlabeled RANTES competed specific binding of 125I-labeled RANTES to monocytes, iDC or mDC with IC50 values of 7.5, 1.0 or 8.9 nM respectively. On the other hand, the specific RANTES-binding sites of iDC and mDC were about 180,000, 420,000 or 160,000 sites/cells respectively. The IC50 values and RANTES-binding sites of monocytes were similar to the previously reported values (50). These results indicate that the binding of RANTES to iDC was higher than to monocytes or mDC, and that monocytes and mDC exhibited similar levels of binding for RANTES.



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  		Fig. 4. Inhibition by RANTES of [125I]RANTES binding to monocytes, iDC and mDC. [125I]RANTES (~130,000 c.p.m.) was added into each tube of cell suspension in the presence or absence of increasing concentrations (0.001–1000 nM) of unlabeled RANTES and the culture was incubated for 1 h at room temperature. The IC50 values and the specific binding sites cells were calculated as described in Methods. The results are representative of three experiments performed in duplicate with similar results.

 
Effect of RANTES and MIP-3ß on the induction of protein tyrosine phosphorylation events in monocytes, iDC and mDC
Engagement of chemokine receptors by respective ligands activates PTK-dependent cascades in various cell types (21–27). As shown in Fig. 5Go(A), stimulation with RANTES caused a tyrosine phosphorylation of intracellular proteins in both monocytes and iDC, while this stimulation caused little or no tyrosine phosphorylation events in mDC. Similar results were observed in these cells stimulated with MIP-1{alpha} and eotaxin (data not shown). On the other hand, stimulation with MIP-3ß failed to induce tyrosine phosphorylation events in both monocytes and iDC, whereas these events were observed in MIP-3ß-stimulated mDC (Fig. 5AGo).



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  		Fig. 5. Chemokine-induced protein tyrosine phosphorylation events in monocytes, iDC and in mDC. (A) Effect of RANTES and MIP-3ß on the induction of tyrosine phosphorylation events in monocytes, iDC and mDC. Monocytes (1–3), iDC (4–6) and mDC (7–9) (4x106) were either unstimulated (1,4 and 9) or incubated with 10 ng/ml of RANTES (2,5 and 78) or MIP-3ß (3,6 and 79) for 5 min at 37°C. (B–C) Effect of RANTES and MIP-3ß on the activation of Src in monocytes, iDC and mDC. Monocytes (1 and 72), iDC (3 and 74) and mDC (5 and 76) (107) were either unstimulated (1,3 and 75), or incubated with 10 ng/ml of RANTES (B) or MIP-3ß (C) (2,4 and 76) for 5 min at 37°C. The total cell lysates (A) and immunoprecipitates with antibodies to Src (60 kDa) (B and C) were fractionated by 12% SDS–PAGE and blotted onto PVDF membranes. The protein samples were analyzed by western blotting with anti-pTyr mAb (phospho-proteins) (A–C) or indicated antibodies to ensure similar amounts of protein in each sample (B and C). The results are representative of 10 experiments with similar results.

 
Src-family PTK are activated in response to stimulation of a variety of GPCR and are necessary for linking GPCR with MAPK activation (30). We therefore examined the influence of RANTES and MIP-3ß on the activation of Src in monocytes, iDC and mDC. Ligation by RANTES increased tyrosine phosphorylation of Src in monocytes and iDC, while this stimulation failed to cause tyrosine phosphorylation of this molecule in mDC (Fig. 5BGo). Conversely, MIP-3ß caused tyrosine phosphorylation of Src in mDC, but not in monocytes and iDC (Fig. 5CGo).

To address the role of CCR in the initiation of chemokine-induced tyrosine phosphorylation evens, we examined tyrosine phosphorylation of CCR-1, -3 and -5 following stimulation with RANTES in monocytes, iDC and mDC. Stimulation with RANTES induced tyrosine phosphorylation of CCR-1, -3 and -5 (Fig. 6A–CGo) in monocytes and iDC, whereas this stimulation caused a slight tyrosine phosphorylation of these CCR in mDC (Fig. 6A–CGo).



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  		Fig. 6. Effect of RANTES on the tyrosine phosphorylation of CCR-1, -3 and -5 in monocytes, iDC and in mDC. Monocytes (1 and 2), iDC (3 and 4) and mDC (5 and 6) (107) were either unstimulated (1,3 and 5) or incubated (2,4 and 6) with 10 ng/ml of RANTES for 5 min at 37°C. Immunoprecipitates with antibodies to CCR-1 (41 kDa) (A), CCR-3 (41 kDa) (B) or CCR-5 (38 kDa) (C) were fractionated by 12% SDS–PAGE and blotted onto PVDF membranes. The protein samples were analyzed by Western blotting with anti-pTyr mAb (phospho-proteins) or indicated antibodies to ensure similar amounts of protein in each sample. The results are representative of 10 experiments with similar results.

 
p125FAK and Pyk2 are phosphorylated and activated by RANTES stimulation of monocytes and iDC, but not of mDC
To address the molecular events responsible for the reduction of RANTES-induced chemotaxis of mDC, the activation states of p125FAK and Pyk2 were examined (Fig. 7A and BGo). Engagement by RANTES induced significant tyrosine phosphorylation and enzymatic activation of p125FAK and Pyk2 in monocytes and iDC, but only slight activation in mDC.



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  		Fig. 7. RANTES induces activation of p125FAK and Pyk2 in monocytes and iDC, but not in mDC. Monocytes (1 and 2), iDC (3 and 4) and mDC (5 and 6) (107) were either unstimulated (1 , 3 and 5) or incubated with RANTES (10 ng/ml) (2 , 4 and 6) for 5 min at 37°C. Immunoprecipitates with antibodies to p125FAK (125 kDa) (A), Pyk2 (112 kDa) (B) or paxillin (68 kDa) (C) were analyzed by Western blotting with anti-pTyr mAb (phospho-proteins) or indicated antibodies to ensure similar amounts of protein in each sample. In parallel experiments, immunoprecipitates with antibodies to p125FAK (A) or Pyk2 (B) were assayed for autophosphorylation or kinase activity with enolase (47 kDa) as a substrate. The results are representative of 10 experiments with similar results.

 
We further examined the effect of RANTES on tyrosine phosphorylation of paxillin, a major cytoskeletal component of focal adhesion. As shown in Fig. 7Go(C), tyrosine phosphorylation of paxillin were observed in monocytes and iDC following stimulation with RANTES although treatment of mDC with RANTES induced lower level of tyrosine phosphorylation of paxillin.

RANTES induces activation of ERK2, SAPK/JNK and p38mapk in monocytes and iDC, but not in mDC
To clarify the potential involvement of ERK2, SAPK/JNK and p38mapk in the regulation of chemotaxis of monocytes, iDC and mDC, the cells were unstimulated or stimulated with RANTES, and the level of tyrosine phosphorylation and kinase activities of the MAPK were examined (Fig. 8A–CGo). Stimulation of monocytes or iDC with RANTES increased tyrosine phosphorylation and kinase activities of ERK2, SAPK/JNK and p38mapk compared to unstimulated cells. On the other hand, the tyrosine phosphorylation of MAPK and their kinase activities in mDC induced by RANTES were lower than those of RANTES-stimulated monocytes and iDC.



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  		Fig. 8. RANTES induces activation of ERK2, SAPK/JNK or p38mapk in monocytes and iDC, but not in mDC. Monocytes (1 and 2), iDC (3 and 4) and mDC (5 and 6) (107) were either unstimulated (1,3 and 5) or incubated with RANTES (10 ng/ml) (2,4 and 6) for 5 min at 37°C. The total cell lysates were analyzed by Western blotting with mAb to ERK2 (42 kDa) (A), SAPK/JNK (46 and 54 kDa) (B), p38mapk (38 kDa) (C) or their tyrosine phosphorylated forms (A–C). In parallel experiments, immunoprecipitates with anti-tyrosine phosphorylated ERK2 (for ERK2 kinase activity) (A), c-Jun fusion protein (for SAPK/JNK kinase activity) (B) or anti-tyrosine phosphorylated p38mapk (for p38mapk kinase activity) (C) were assayed for their kinase activities with Elk-1 (40 kDa) (for ERK2 kinase activity) (A), c-Jun fusion protein (33 and 35 kDa) (for SAPK/JNK kinase activity) (B) or ATF-2 (35 kDa) (for p38mapk kinase activity) (C) used as substrates. The results are representative of 10 experiments with similar results.

 

   Discussion
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
References
 
Responsiveness of DC to CC chemokines is associated with their maturation (19,40,41,47). However, the mechanism underlying these evens remains unclear. Our findings show that expression levels of chemokine receptors (CCR-1, -3, -5 and -7) as well as their downstream signaling events regulate chemotaxis of human monocyte-derived DC.

Transcriptional and cell-surface expression of CCR-1 and -5 were previously shown in various types of human DC (19,38–44). However, there are conflicting reports about the expression of CCR-3 in these cells (38–43). In accordance with previous reports (39,42,43), we showed that iDC constitutively expressed the transcripts and products on the cell surface of CCR-3 as well as CCR-1 and -5 (Fig. 1B and CGo). Furthermore, we observed that iDC migrated to their respective ligands such as RANTES (for CCR-1, -3 and -5), MIP-1{alpha} (for CCR-1 and -5) and eotaxin (for CCR-3) (Figs 2 and 3GoGo), and the migratory response to these chemokines by these cells were chemotactic and not due to chemokinesis because migration was not observed in the absence of a chemokine gradient (47). On the other hand, Chan et al. (43) have recently reported that the transcript of CCR-6 was constitutively expressed in monocyte-derived iDC, while its cell-surface expression was undetectable in these cells. We (47) have also recently reported that the transcriptional expression of CCR-6, but not its cell-surface expression, was detected in iDC, and these cells failed to migrate to MIP-3{alpha}. These results suggest that CCR-1, -3 and -5, but not CCR-6, may be functionally expressed on the cell surfaces of monocyte-derived iDC.

Previous studies have shown that transcription and cell-surface expression of CCR were undetectable in mDC (40,53,54). We showed that little or no transcriptional expression of CCR-1, -3 and -5 were observed in mDC (Fig. 1CGo). Furthermore, their cell-surface expressions were still detectable in these cells, although these expression levels were significantly reduced in mDC as compared with those of iDC (Fig. 1BGo) (47). This discrepancy may be due to the binding affinities of mAb for the respective CCR or the preparation of mDC because the expression levels of CCR in iDC in our system (46,47) were higher than those of previous reports (52,53). Alternatively, we also showed that the levels of transcriptional expression of CCR were associated with, but not completely paralleled with, the levels of their cell-surface expression in the development of monocyte-derived DC (Fig. 1B and CGo). Recent studies have shown that PTK-dependent cascades and metalloproteinases contributes to the regulation of cell-surface expression of chemokine receptors (55). Thus, other molecular mechanism(s) as well as the transcriptional regulation may be involved in the reduced cell-surface expressions of these CCR in the development of monocyte-derived DC.

Treatment of monocytes, iDC and mDC to CC chemokines with genistein and pertusis toxin suppressed their chemotactic responses to CC chemokines (Fig. 3Go). Furthermore, stimulation of monocytes and iDC with RANTES caused activation of PTK-dependent cascades including Src-, FAK- and MAPK-mediated signaling events, while this stimulation resulted in the lower activation of these events in mDC as compared with those of monocytes and iDC (Figs 5–8GoGo). Conversely, MIP-3ß caused a tyrosine phosphorylation of several intracellular proteins, including Src, in mDC, whereas this stimulation failed to induce these events in monocytes and iDC (Fig. 5Go). These results suggest that Gi-coupled receptor-mediated PTK-dependent cascades may directly regulate the chemotaxis of monocytes, iDC and mDC to the respective chemokines.

We showed that mDC exhibited lower cell-surface expression levels of CCR-1, -3 and -5 than those of iDC, and these expression levels were similar to those of monocytes (Fig. 1BGo). Furthermore, the abilities of monocytes as well as iDC to migrate to inflammatory chemokines were more potent than those of mDC (Figs 2 and 3GoGo). These results indicate that the changes in the chemotactic migratory responses for inflammatory chemokines during development of monocyte-derived DC are not simply regulated by their cell-surface expression levels of CCR. Although the down-regulation of cell-surface expression of these CCR may partly contribute to the defective chemotactic migratory properties of mDC for inflammatory chemokines, other molecular mechanism may be also involved in this phenomenon. On the other hand, the level of the transcriptional expression of CCR-7 is paralleled with the responsiveness to MIP-3ß (Figs 1–3GoGo). Therefore, the expression level of CCR-7 may directly reflect the ability to migrate to MIP-3ß, although the cell-surface expression level of CCR-7 remains unclear.

We showed that the chemotactic migratory response of iDC to RANTES was higher than that of monocytes by ~2-fold (Figs 2 and 3GoGo). On the other hand, RANTES-induced tyrosine phosphorylation of intracellular proteins, including CCR-1, -3 and -5 as well as Src in iDC was higher than those of RANTES-stimulated monocytes (Figs 5 and 6GoGo), while the degree of activation of FAK, Pyk2 and paxillin as well as MAPK in iDC was almost similar to those of monocytes following stimulation with RANTES (Figs 7 and 8GoGo). The reason why the degree of activation states of FAK, Pyk2 and paxillin as well as MAPK did not reflect the difference of the responsiveness between monocytes and iDC to inflammatory chemokines remains unclear. However, several possibilities may account for these phenomena. Chemokine responsiveness of monocytes to inflammatory chemokines may be potentially lower than that of iDC. Indeed, the maximal chemotactic migration of monocytes was observed at a concentration of 100 ng/ml of inflammatory chemokines (e.g. RANTES) and this response was gradually decreased when further increased concentrations of chemokines were used for chemoattractants (data not shown). The reason why maximal chemokine response of monocytes was lower than that of iDC may be due to the their lower ability to organize the cytoskelton in response to chemokines because iDC is known to possess a developed cytoskeletal system and show a morphologically more complexed form (dendritic morphology) (56,57). Therefore, chemokine responses of monocytes and iDC are different at the same concentrations of chemokines even though a similar degree of activation of these regulatory molecules was observed between monocytes and iDC. Alternatively, the tyrosine phosphorylation states of Src as well as CCR in monocytes was lower than those of iDC following stimulation with RANTES (Figs 5B and 6A–CGoGo). Therefore, the activation of early signaling events involving Src-dependent cascades may be responsible for the difference of the responsiveness between monocytes and iDC to inflammatory chemokines, although ligand-induced activation of FAK, Pyk2 and paxillin as well as MAPK may also contribute to their chemotactic responses.

The molecular mechanisms underlying the reduction in the signaling capacity of CCR in response to inflammatory chemokines in mDC, in terms of the defective activation of PTK-dependent cascades, remain unclear. We showed that stimulation of monocytes with RANTES induced activation of CCR-mediated downstream signaling events (Figs 5–8GoGo), suggesting that their cell-surface expression levels of CCR may be sufficient to initiate these molecular events. In contrast, RANTES induced lower activation of CCR-mediated PTK-dependent cascades in mDC (Figs 5–8GoGo), although monocytes and mDC express similar levels of CCR on their surface (Fig. 1BGo). We also observed that monocytes and mDC exhibited similar binding for RANTES (Fig. 4Go), suggesting that these cell types may possess similar binding capacities for RANTES via CCR-1, -3 and -5, although these binding capacities may be lower than those of iDC. On the other hand, we demonstrated that RANTES induced tyrosine phosphorylation of CCR-1, -3 and -5 in monocytes as well as iDC, while its stimulation resulted in little or no tyrosine phosphorylation of these CCR (Fig. 6A–CGo), indicating that early section of CCR-mediated signaling was suppressed in mDC. Hashimoto et al. (58) have previously reported that the development of monocyte-derived DC is accompanied with an increase or decrease of expression or availability of various molecules. It has been shown that the tyrosine phosphorylation states of the targeted proteins are regulated by their respective kinases and phosphatases (59–61). We (59) and other (60,61) have previously suggested that a family of protein tyrosine phosphatases may exist to antagonize a large number of kinases and these phosphatases may be negatively involved in signaling in certain cells. Although we failed to detect association of CCR with any protein tyrosine phosphatases and inositol phosphatase, including Src homology 2-containing protein tyrosine phosphatases (SHP)-1, SHP-2 and Src homology 2-containing inositol phosphatase (SHIP) (data not shown), CCR-mediated signaling involving PTK-dependent cascades may be repressed by unknown or identified regulatory molecule(s), which are specifically expressed during the maturation of DC. Further study will be needed to test such possibilities.

In summary, our results provide a possible mechanism for the chemotactic migratory capacity of DC in response CC chemokines involving the cell-surface expressions of CCR and their downstream PTK-dependent signaling events. Chemokines and their respective receptors play important roles in inflammatory and allergic diseases as well as immune responses (18). In addition, DC are critically involved in autoimmune diseases, graft rejection and virus infection (1–4). On the other hand, the effective targeting of tumor antigen-loading DC into tumor tissues or lymph nodes around these sites has been shown to be useful for tumor immunotherapy (62,63). Defining the precise mechanisms of the chemotactic migratory capacity of DC may provide further insights into the role of these cells in immune-related diseases and facilitate the use of DC in vaccinations for cancer treatment.


   Acknowledgments
 
We would like to thank Dr K. Takatsu and Dr S. Takaki for analysis of the binding data by Ligand, and Mrs M. Midorikawa and Mrs M. Toda for secretarial assistance.


   Abbreviations
 
APC antigen-presenting cell
ATF activating transcription factor
CCR CC chemokine receptor
CXCR CXC chemokine receptor
DC dendritic cell
ECL enhanced chemiluminescence
ERK extracellular signal-regulated kinase
FAK focal adhesion kinase
GPCR G protein-coupled receptor
GM-CSF granulocyte-macrophage colony-stimulating factor
HRP horse radish peroxidase
iDC immature DC
Jak Janus kinase
LPS lipopolysaccharide
MAPK mitogen activated protein kinase
mDC mature DC
MIP macrophage inflammatory protein
PBMC peripheral blood mononuclear cell
PTK protein tyrosine kinase
PI propidium iodide
pTyr phosphotyrosine
RANTES regulated on activation normal T cell expressed and secreted
SAPK/JNK stress-activated protein kinase/c-Jun N-terminal kinase
SHIP Src homology 2-containing inositol phosphatase
SHP Src homology 2-containing protein tyrosine phosphatase
Stat signal transducer and activator of transcription
TNF tumor necrosis factor

   Notes
 
Transmitting editor: M. Miyasaka

Received 10 July 2000, accepted 24 October 2000.


   References
Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 References
 

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